The search for improved therapeutic agents for the treatment of inflammatory, as well as fibrotic diseases, has received much attention in recent years. Neonatal Respiratory Distress Syndrome (RDS), a lung surfactant deficiency disease, is a condition of particular interest in that it is one of the major causes of mortality in premature neonates. While introduction of surfactant therapy dramatically improves survival of RDS patients, the development of chronic inflammatory and fibrotic disease in a significant percentage of this patient population is a major problem. Likewise, hereditary fibronectin-deposit glomerular nephropathy leads to end stage renal failure when patients' kidneys become blocked and no longer filter the blood. Hereditary glomerular nephropathy is characterized by fibronectin deposits and fibrosis of the kidneys. In both diseases, fibronectin deposition and fibrosis render the organ non-functional, and eventually, unable to support life. Thus, these patients require chronic hemodialysis or kidney transplantation.
PLA.sub.2 is one of the enzymes responsible for hydrolysis of the surfactant phospholipids. Human uteroglobin, also known as "CC10", inhibits the activity of phospholipase A.sub.2 (PLA.sub.2) in vitro [Levin, S. W., et al. Uteroglobin inhibits phospholipase A.sub.2 activity. Life Sci. 38:1813-1819 (1986); Singh, G. et al. Clara Cell 10 kda protein (CC10): Comparison of Structure and Function to Uteroglobin. Biochem. Biophys. Acta. 1039:348-355 (1990); Mantile, G. et al Human Clara Cell 10 kDa Protein Is The Counterpart of Rabbit Uteroglobin. J. Biol. Chem. 268:20343-20351 (1993)]. Human uteroglobin was first isolated in 1988 as a secretion product from the Clara cells of the lungs (Singh, G., et al. Identification, Cellular Localization, Isolation and Characterization of Human Clara Cell-Specific 10 kD Protein. J. Histochem. Cytochem., 36:73-80 (1987). (The designation "CC10" was derived from "Clara cell 10 kDa".). It is a small globular homodimeric protein, and migrates in electrophoretic gels at a size corresponding to 10 kDa (Singh, G., et al. Identification, Cellular Localization, Isolation and Characterization of Human Clara Cell-Specific 10 kD Protein. J. Histochem.Cytochem., 36:73-80 (1987). (The designation "CC10" was derived from "Clara cell 10 kDa"). Human uteroglobin (hUG) is abundant in the adult human lung, and comprises up to about 7% of the total soluble protein (Bernard, A. et al, Protein 1 is a Secretory Protein of the Respiratory and Urogenital tracts identical to the Clara Cell Proteins. Clin. Chem. 38:434-435 (1992)). However, its expression is not fully activated in developing human fetus until the last few weeks of gestation. Consequently, the extracellular lung fluids of pre-term infants contain less human UG than those of adults, and may be deficient in these neonates (Dhanireddy, R., El-Ali, M., Murty, L., and A. B. Mukheijee, Pediatric Research 23:463A (1988) and Dhanireddy, R., Lim, M., and A. B. Mukheijee, Pediatric Research 33:323A (1993)).
PLA.sub.2 s are a class of endogenous enzymes that hydrolyze the sn2 position ester bond of glycerophospholipids. They play critical roles in the inflammatory response because they release arachidonic acid (AA) from cellular phospholipid reservoirs. AA is metabolized to a number of potent inflammatory mediators in a process referred to as the arachidonic acid cascade (Piomelli, D. Arachidonic Acid in Cell Signaling. Op. In Cell Biol. 5:274-280 (1993)). Several acute and chronic clinical conditions have been characterized with elevated serum or local PLA.sub.2 activity (see Table below). Native rabbit uteroglobin (UG), discovered in 1967 and purified from uteri of pregnant rabbits (Krishnan, R. S. and Daniel, J. C., Jr. "Blastokinin: Inducer and Regulator of Blastocyst Development in the Rabbit Uterus." Science 158:490-492 (1967); Beier H. M. Verhandl Deut. Zool. Ges., Heidelberg (1968)), inhibits PLA.sub.2 activity in vitro (Levin, 1988; supra). Clinical conditions in which elevated PLA.sub.2 activity is documented are set forth in the following Table.
TABLE Diseases Sites Rheumatoid arthritis Serum, synovial fluid, WBC Collagen vascular diseases Serum Pancreatitis Serum Peritonitis Peritoneal fluid and cells Septic shock Serum ARDS.sup.a Serum and alveolar fluid Acute renal failure Serum Autoimmune uveitis Serum, aqueous humor Bronchial asthma Bronchial fluid .sup.a Adult respiratory distress syndrome (Modified from Mukherjee et al, 1992)
Amino acid analysis of purified human UG reveals that it is structurally similar to rabbit UG but not identical. 37 of 70 amino acids are identical between human and rabbit UG (see FIG. 1). The "UG-like" proteins, including human UG/CC10, rat CC10, mouse CC10 and rabbit UG, exhibit species-specific and tissue-specific antigenic differences, as well as differences in their tissue distribution and biochemical activities in vitro. UG-like proteins have been described in many different contexts with regard to tissue and species of origin, and have also been identified using numerous different names, including uteroglobin, blastokinin, CC10 kDa protein (from rat lung), urine-protein 1 or "P1" (human), progesterone-binding protein (rabbit uterus), PCB-binding protein (rat lung), and CC16 (human lung), which has created confusion in the literature. At present there are no known physiological roles for these proteins (Umland, T. C., et al. Structure of a Human Clara Cell phospholipid-binding protein-ligand, complex at 1.9 Aresolution, Nature Struct. Biol. 1:538-545 (1994); Hard, T. et al, Solution Structure of Mammalian PCB-binding Protein in Complex with a PCB. Nat. Struct. Biol. 2:983-989 (1995); Umland, T. C. and M. Sax. Twixt form and function. Nat. Struct. Biol. 2:919-922 (1995); Stripp, B. R., et al. Clara cell secretory protein: a determinant of PCB bioaccumulation in mammals. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L656-L664 (1996).
The absence of structural identity among UG-like proteins makes it impossible to predict whether a protein will possess in vivo function based on in vitro or other activity exhibited by a structurally related protein in the context of an human therapeutic. For example, human uteroglobin binds less than 5% of the amount of progesterone than rabbit UG binds in the same assay (Singh, G., et al. Clara Cell 10 kDa Protein (CC10): Comparison of Structure and Function to Uteroglobin. Biochem Biophys Acta 1039:348-355 (1990). Human UG has a lower isoelectric point (4.6) than rabbit UG (5.4). Moreover, a UG knockout mouse recently generated to eliminate expression of UG has Clara cells which exhibit odd intracellular structures in place of UG secretion granules, but there is no other phenotype. This observation is highly significant since the anticipated phenotype was inefficient pulmonary finction accompanied by pulmonary inflammation and fibrosis. The knockout mouse shows no evidence of pulmonary impairment or abnormality, indicating that the UG protein has no significant role in surfactant homeostasis as had been previously suggested (Lesur, O., et al. Clara Cell Protein (CC16) Induces a Phospholipase A.sub.2 -mediated Inhibition of Fibroblast Migration in Vitro. Am. J. Respir. Crit. Care Med. 152:290-297 (1995); Umland, T. C., et al, 1994; supra).
Despite years of studies and accumulation of data on UG-like proteins, the biological roles of these proteins and their inhibitory effect in vivo on PLA.sub.2 s and the inflammatory process remains unclear. There are no effective PLA.sub.2 inhibitors presently available for clinical use. To date, only a few PLA.sub.2 inhibitors have progressed into clinical trials, but none have qualified for commercial marketing.
Fibronectin (Fn) is a 200 kDa glycoprotein which exists in several different forms and is secreted by different tissues. Fn is an essential protein and attempts to generate knockout mice showed that it has a central role in embryogenesis. Fn also plays a key role in inflammation, cell adhesion, tissue repair and fibrosis, and is deposited at the site of injury. Plasma fibronectin, (pFn) is secreted by the liver and circulates in the plasma. In the lung, cellular Fn (cFn) is secreted upon inflammation and injury. Both types of Fn are chemotactic factors for inflammatory cells and fibroblasts. Large numbers of inflammatory cells (neutrophils) and fibroblasts infiltrate the lung during inflammatory episodes, which can lead to pulmonary fibrosis and ultimately death. Elevated levels of Fn have been detected in human clinical conditions such as neonatal RDS and BPD of the lung, and glomerular nephropathy of the kidney.
An urgent need exists for an effective agent for treating inflammatory conditions, particularly RDS, and fibrotic diseases where fibronectin deposition is a causative factor, which does not cause deleterious side-effects. The present invention discloses novel physiological functions of UG and seeks to fill that need.